Our long range goal continues to be a detailed understanding of the molecular mechanisms involved in site-specific recombination, including the associated higher-order structures and protein-DNA and protein-protein interactions. As a model we are studying the Int-dependent recombination pathways of the lambdoid phages lambda, phi80, and P22. The major portion of our effort is directed toward the A system but the information we have been obtaining about the other two systems is proving to be very useful at both the experimental and conceptual levels. The experiments proposed in this application are intended to complement studies being carried out in other laboratories. They are based primarily on the following three groups of results we obtained during the previous project period: a) the Int protein has a unique dual DNA recognition capacity that is specified by two autonomous domains and a topoisomerase active site that is highly conserved in more than 15 other proteins; b) there is an additional host-encoded accessory protein (FIS) that ties lambda recombination to cellular physiology and functions, along with the other accessory proteins (IHF and Xis), by inducing very extreme bends in DNA; c) there are numerous and complex interactions among the four kinds of proteins bound at the 15 binding sites on att DNA. Four specific questions are addressed in this proposal: 1)What structural features of the two-domain Int protein are critical for its function as a site-specific recombinase? 2)How do the recombinogenic structures and mechanisms utilize the accessory proteins IHF, FIS and Xis? 3)What are the short-range and long-range interactions that define the higher order structure of the recombinogenic complexes and determine the efficiency and specificity of recombination? 4)How is the extreme directionality of recombination achieved?